THE STETH VOLUME 6, 2012 ISSN: 2094-5906

Larvicidal activity of four Philippine plants against Dengue virus vector

Aedes aegypti(Linn.)

Student Researchers: Lee Marvin C. De Villa, Mary Joy A. Abantes,

Merlina C. Asi, Noelyn Joy C. Balmeo, Alyssa Monique D. Bustillo,
Eunice M. Calangi&Lhuvie Jean R. Cruzado

Faculty Researchers: Oliver Shane R. Dumaoal, RMT &

Carina R. Magbojos, RMT

Abstract -Bioactive compounds, such as the plant-derived

allelochemicals, are of growing interest to the scientific community due to their
known toxicity against several obnoxious vectors of pervasive diseases like
dengue and malaria. Moreover, the development of resistance to synthetic
insecticides has diverted the interest of researchers towards insecticides of plant
origin. Hence, four Philippine plant species, Citrus microcarpa (Calamansi),
Chromolaena odorata (Hagonoy), Nephelium lappaceum (Rambutan), and
Jasminum sambac (Sampaguita) of the respective plant families Rutaceae,
Asteriaceae, Sapindaceae and Oleaceae were evaluated of their larvicidal
activities. The secondary metabolites tannin and citrus, both of which have
shown larvicidal and insecticidal activities, have been found in plant families
Sapindaceae and Rutaceae. Likewise, Jasminum sambac and Chromolaena
odorata have been reported in the literature to contain terpenoids and
organophosphates, two major constituents of agricultural pesticides both
recognized for the potency. The larvicidal activities of ethanolic extracts in three
concentrations (100, 200, 500ppm) from four plants were evaluated against third
instar larvae of dengue mosquito, Aedes aegypti in the laboratory. Their activities
were compared with black pepper, Piper nigrum (positive control) and the
untreated control (distilled water and DMSO). Among the plants bioassayed, the
500 ppm ethanolic extract from C. microcarpa provided 24-hr mortality of 80%,
which is slightly lower than the 100% mortality obtained in black pepper. Multiple
regression analysis also revealed statistically significant relationship (p-value
<0.05) between concentration of the extracts of four plants and mortality rate.
Linearity between larval mortality and exposure time is most significant at 500
ppm concentration with a fitted regression model: % mortality = 16.16x – 25.6 X
time interval (R2=0.944). Endpoint analysis of log-probit transformed curve of
Citrus microcarpa indicates LC50 and LC90 of 451 and 628 mg/l (500 ppm)
respectively. Since C. microcarpa is much cheaper than black pepper, more in
depth study should be done in enhancing its activity by slightly increasing its
concentration or through the addition of synergist and combination with other
potent botanical insecticides. Moreover, the use of C. microcarpa is sage to
humans and other non-target organisms and is an environment-friendly method
of controlling dengue mosquito wrigglers.

Keywords- bioactive compounds, Aedes aegypti, Citrus microcarpa,

Chromolaena odorata, Nephelium lappaceum, Jasminum sambac, larvicidal
activity

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INTRODUCTION

Dengue virus (DV) (genus Flavivirus), the causative agent of dengue

fever (DF) and dengue hemorrhagic fever (DHF) consists four distinct serotypes
(DEN-1, DEN-2, DEN-3, DEN-4) all capable of attaining full spectrum of the
disease (Li et al., 2011) resulting to about 25,000 annual deaths worldwide
(World Health Organization (WHO), 2011). The disease burden emerged as one
of the major public health concerns especially in Southeast Asia where climate,
urban sprawl and deteriorating environmental conditions all promote an easy
spread of the virus (Ooi and Gubler, 2008). WHO (2009) further links the same
factors for a 30-fold increase in the global incidence rate within the last 50 years.
Moreover, the substantial growth in international travels and climate change
exacerbates the problem (Hales et al., 2002; Massad and Wilder-Smith, 2009). In
2011, over 70,000 cases with <1% case-fatality rate has been reported in the
Philippines, more than 20% of which came from National Capital Region alone
(Department of Health (DOH), 2011).

Predation by insect herbivores in their natural habitat drives the positive

evolution of plants where better defenses through production of secondary
metabolites (allelochemicals), are made to ensure species survival (Metlen et al.,
2009). This natural interaction constitutes host-plant resistance and is a function
of biologically synthesized chemicals including alkaloids, cyanogenic glycosides,
terpenoids, phenolics, organophosphates and many others (de F. Fernandes et
al., 2005; Davou and Matur, 2007; Lucia et al., 2008; Morais et al., 2011). Black
et al. (2008) claimed these antiherbivory compounds as prototypes for most
plant-derived mosquito-control agents with spectrum even encompassing
larvicidal and antimicrobial actions.

Although several chemical synthetics against dengue virus vectors have

been made commercially available, reports of resistance from these conventional
controls have been increasing. Recently, knockdown resistance that gave
emergence to two pyrethroid-resistant Culex spp. has been reported in Northern
China and Sri Lanka (Song et al., 2007; Wondji et al., 2008). Moreover,
researchers in South Carolina and India have also claimed that exploitations of
synthetic pesticides on the environment produce detrimental effects to both
aquatic and terrestrial food webs (Bhattacharya and Kaviraj, 2008; Williamson et
al., 2009). This declining efficacy of commercial insecticides have limited its
availability for public use, while those remaining efficient remains beyond the
financial capacity of most developing countries including the Philippines. This
reality makes the finding of eco-friendly, cheaper and biodegradable alternatives
an imperative and important public health goal.

In recent years, development of vaccine against dengue has been the

focal point of research (Durbin et al., 2005; Melino and Paci, 2007; Whitehead et
al., 2007). While in fact several clinical trials of tetravalent vaccine by some
pharmaceutical firms (Acambis, GSK Biological, Hawaii Biotech, InViragen,

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Sanofi Pasteur, Shanta Biotechnics) and government health agencies (FDA,

WRAIR) have begun (Hombach, 2007), the use of botanical bioactive
compounds remains the prevailing model to curtail disease transmission. Farrar
and Whitehorn (2010) further stress their significance as useful adjuncts to future
vaccines with marked synergistic effects between the two. Biological control that
utilizes Cry and Cyt toxins of Bacillus thuringiensis israelensis (Bti), is another
novelty found to be both effective and efficient in killing larvae of many Dipterans.
However, emergence of Bti-resistant Culex spp. (Wirth et al., 2010) and adverse
trophic effects on breeding birds have been noted (Poulin et al., 2010).

While several studies have already proven the efficacy of botanical

bioactive compounds (Jang et al., 2002; Cavalcanti et al., 2004; Pushpanathan et
al., 2006; Jung and Moon, 2011; Lima et al., 2011; Medeiros et al., 2011;
Raghavendra et al., 2011), studies that investigate the larvicidal potentiality of
Philippine plants are lacking. Thus, the ethanolic extracts prepared in various
concentrations (100, 200, and 500ppm) were used against the laboratory-reared
early fourth instar Aedes aegypti larvae (Diptera: Culicidae), the major mosquito
vector of dengue virus in the country.

MATERIALS AND METHOD

Insects

Second-instar larvae of the laboratory-reared A. aegypti were provided

by Dr. Pio A. Javier, Research Professor, Crop Protection Cluster, College of
Agriculture, University of the Philippines Los Banos, College, Laguna. The
obtained larvae were reared at polyene plastic containers (12 x 6) filled with 300
ml dechlorinated aged tap water and aerated through lid punctures. They were
fed intermittently once a day with brewers yeast and flake fish food (30% to 40%
protein) with increasing amounts for successive instars. Formation of residual
food artifacts detrimental to the larvae development was closely monitored and
periodically removed. This prohibited scum formation (biofilm) that deprives
larvae of atmospheric oxygen (Asahina, 1964). Optimal conditions were
sustained at 25 ± 2 °C with 70 ± 5% relative humidity (RH), and a photoperiod of
16 : 8 (Light : Dark) h (Jang, et.al., 2002).

Plant Materials

The peels (exocarp) of Citrus microcarpa, seeds of Nephelium

lappaceum, flowers of Jasminum sambac and leaves of Chromolaena odorata
were gathered from the parent plants during the flowering and fruiting seasons at
separate locations in Batangas City (Table 1). These respective parts
represented the highest concentrations and yield of bioactive compounds
(Amusan et al., 2005; Ragasa et al., 2005; Joy and Raja 2008). Dr. William
Gruezo of the College of Arts and Sciences, Institute of Biological Sciences,
UPLB, taxonomically identified the plants.

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TABLE 1
General information of four Philippine plants tested
Plant Species Family Part Yield*(% wt/wt)
Collected

Citrus microcarpa Rutaceae Peel 12.77

Chromolaena odorata Asteriaceae Leaf 6.60

Jasminum sambac Oleaceae Flower 5.00

Nephelium lappaceum Sapindaceae Seed 6.93

*
Yield (%) = (Dried weight of ethanol extract\dried weight of sample) x 100

Preparation of Stock Solution

The samples were washed three times with 500 ml distilled water to
remove impurities,
Table 1shade-dried and
General information finely
of four powdered.
Philippine plants tested Each sample (150 grams
weighed through analytical balance) was extracted twice with 300 ml ethanol
(boiling point 78°C) in an airtight bottle at room temperature (26 + 3 °C) for three
days and filtered. The combined filtrates (ethanolic extract) were then
concentrated into a semisolid state for three days under laminar hood. The
residues were weighed and made into stock solution (10% w/v) using distilled
water as a solvent and 1% dimethyl sulfoxide (DMSO) as penetrating agent.
Stock solution was kept inside the refrigerator set at 2-6 °C until use.

Larvicidal Bioassays

The larvicidal activities of the different ethanol plant extracts were

a
bioassayed Yield
against third
(%) = (Dried weightinstar
of ethanollarvae ofweight
extract\dried Aedes aegypti
of sample) x 100 following the WHO
standard protocols (WHO, 2005) with slight modifications. A 10% stock solution
was prepared for each ethanol plant extract using 1% dimethyl sulfoxide
(DMSO). Three concentrations (100, 200, and 500 ppm) of each of the ethanol
extract was prepared by adding the required volume of stock solution in 50 ml of
distilled water using plastic cups (100 ml). In each plastic cup, 15 A. aegypti third
instar larvae were introduced. A separate control set-up using 1% DMSO and
distilled water was used in each experiment. Meanwhile, black pepper (piper
nigrum) concentrate was utilized as the positive control. The treated and control
(positive and negative) larvae were held at same conditions. All treatments were
replicated three times. Mortality of wrigglers was monitored starting at 0.5, 1, 2,
3, 4, 14, 28 and 72 hrs after exposure to the ethanol extract during which one
gram of yeast was added and conditions maintained 25 + 2 °C. Larvae were
considered dead when they fail to move following tapping with separate
applicator sticks for a minimum of five times (Lucia et al., 2008).

Data analysis

The effectiveness of the ethanolic extracts from different plants was

determined by regression analysis of the log-probit transformed linear curve

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using SPSSv18® (Finney, 1981). For Citrus microcarpa, toxicity was further
evaluated using the median lethal concentration (LC50) and LC90 obtained from
the regression line (see Table 4). For the remaining plants, Nephelium
lappaceum, Chromolaena odorata and Jasminum sambac, LC50 and LC90 could
not be reliably computed because the highest mortality was lower than 27%
(recommended mortality should be close to 10 to 90%), besides, their regression
line is statistically incompatible for LC derivation.

RESULTS AND DISCUSSION

Ethanolic extract

The percent yield (Yield % wt/wt) for each ethanol extract is provided in
Table 1. As seen in the table, Calamansi (C. microcarpa) provided the highest
yield of 12.77% whereas Sampaguita (J. sambac) gave the lowest yield of 5. The
degree of pigmentation of the plants does not correlate with amount yield.
Solubility of the compounds with ethanol may explain the variability but other
contributing factors must also be taken into consideration (e.g. plant part).

Statistical Findings

Scheffe’s test revealed statistically significant relationship (p-value <0.05)

between the mortality rate and the species of plants bioassayed using three
different concentrations (100, 200, 500 ppm). The dose-mortality relations for
100, 200 and 500 ppm have corresponding p-values of 0.041, 0.104 and 0.037.
This indicates that dose-mortality relationship is most significant at 500 ppm
concentration (p-value 0.037) with a confidence interval of 96.3%. This high p-
value implies that mortalities tend to improve with higher extract concentrations
and thus is most predictable at 500ppm. Although mortality at 500ppm is not
solely attributed to the plant extract, it is the concentration to which the plant
extract as a factor of larval mortality was most profound (data not shown).

Figure 1

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Relationship of % mortality of Aedes aegypti larvae against different ethanolic

concentrations (100ppm, 200ppm and 500ppm) of the five treated plants

The data on percent mortality of A. aegypti due to different

concentrations of five plant materials during the seven different time intervals are
provided in Table 2. On the other hand, the relationship between % mortality and
ethanolic plant extract can easily be gleaned in figure 1. Among the test plants,
only C. microcarpa provided significantly highest toxicity against the 3rd-instar
larvae of Aedes aegypti. As early as 30 mins of exposure to 500 ppm, 2%
mortality was observed (figure 2). This further increases to 80% after 24 hours
whereas for 100 and 200ppm, mortalities were not observed until 2-hour post
exposure. There was no significant difference between the 24-hour and 72-hour
mortality rates in which 91.3% of the larvae died at 500 ppm. The ethanolic
extract of Piper nigrum, the positive control used in the experiment, provided
100% mortality as early as 4 hr after exposure to 500 ppm. The same mortality
rates were observed after 24 hour of exposure to 100 and 200ppm. This results
collaborates with the reports regarding the effectiveness of black pepper against
the wrigglers of A. aegypti (Bhuyan and Guswami, 2006; Vasudevan et al., 2009).
Further increase in mortality rate of C. microcarpa was most dramatic between 4
and 24-hour intervals (51.3-80%). The remaining plants (C. odorata, J. sambac
and N. lappaceum) displayed the similar inclinations, (Figure 2) however the
mortality rate of <20% deemed it insignificant. No mortality was obtained from the
controls until the 72-hour exposure confirming the non-toxicity of distilled water
and 1% DMSO to the larvae (Figures 1 and 2). Moreover, this indicates that the
wrigglers were properly handled such that the mortalities observed when
exposed to the ethanolic plant extracts are primarily due solely to the activity of
the extracts.

Figure 2
Relationship between Aedes aegypti larval mortality and time intervals.
Each point represents the mean of three replicates of the four Philippin plants

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and their corresponding mortalities at 200ppm

On the other hand, the ethanolic extracts of the remaining three plants,
Chromolaena odorata, Jasminum sambac and Nephelium lappaceum caused
mortality lower than 9% even at very high concentration of 500 ppm (Table 2)
suggesting that these plants in the absence of synergist or any potentiators have
no potential activity for the control of Aedes aegypti larvae.

TABLE 2
Percentage mortality in time of five Philippine plant ethanolic extracts
rd
on 15 A. aegypti 3 instar larvae in three replicates

0.5hr 1hr 2hrs 3hrs 4hrs 24hrs 72hrs

Concentration (ppm)

N % N % N % N % N % N % N %
Citrus
microcarpa 100 0 0 0 0 0.3 2 1 6.7 1.7 11.3 4.7 31.33 7 46.7

200 0 0 0 0 1 6.7 2.7 18 5 33.3 8 53.3 10.7 71.3

500 0.3 2 1 6.7 2 13.3 4.3 28.7 7.7 51.3 12 80 13.7 91.3
Chromolaena
odorata 100 0 0 0 0 0 0 0 0 0 0 0.7 4.7 1.7 11.3

200 0 0 0 0 0 0 0 0 0.3 2 1 6.7 2 13.3

500 0 0 0 0 0.3 2 1 6.7 1 6.7 2 13.3 4 26.7

Jasminum
sambac 100 0 0 0 0 0 0 0 0 0 0 0 0 0.3 2

200 0 0 0 0 0 0 0 0 0 0 0.7 4.7 1.3 8.7

500 0 0 0 0 0 0 0 0 0.3 2 1.3 8.7 2.7 18

Nephelium
lappaceum 100 0 0 0 0 0 0 0 0 0 0 0 0 0.3 2

200 0 0 0 0 0 0 0 0 0 0 0.7 4.7 1.7 11.3

500 0 0 0 0 0 0 0 0 0.3 2 1 6.7 2.7 18

Piper nigrum
100 1 6.7 4 26.7 6 40 8 53.3 10 66.7 15 100 15 100

200 3 20 7 46.7 10 66.7 13 86.7 14 93.3 15 100 15 100

500 7 46.7 10 66.7 12 80 14 93.3 15 100 15 100 15 100

= Number of death recorded; % = % mortality

As can be seen on table 2, there were no significant increases in

mortalities after 72 hours of exposure. It is unlikely that the ethanol extracts of the
three plants accounted for the deaths, however this claim is too premature to be
decisive. The cause of the death may be inherent to the larvae itself as there
were no reliable means to ensure the consistency in vitality of the experimented
larvae. However, the absence of dead wrigglers in the control indicates that the

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larvae are healthy and were properly handled although this does not resolve the
issue of vitality. The accepted mortality rate in the control is 10% but corrected
mortality should be computed as indicated by Finney (1981). If larvae are weak
then majority of them will die in 72 hrs, a finding not observed for the three
plants. All the other variables (temperatures, ventilation, humidity and lightings)
did not have significant deviations during the three-day exposure, ruling them out
as factors for the larval death. Besides, the control set up composed of 50%
distilled water and 1% DMSO exhibited consistent 0% mortality all throughout
(Figure 2).
TABLE 3
Bioequivalence of the lethal concentrations (LC50 and LC90) of the ethanolic
extracts of Citrus microcarpa and Piper nigrum on third instar larvae of Aedes
aegypti
Larvicidal Activity (mg/l)
Ethanolic extract concentrations
LC50 CI 95% LC90 CI 95%

100ppm 757 610 – 1034 1315 977 – 2285

Citrus microcarpa 200ppm 581 481 - 731 1009 788 – 1581
500ppm 451 372 - 548 628 628 – 1151
100ppm 240 175 – 337 891 592 – 1641
P. nigrum 200ppm 124 87-152 406 282-671
500ppm 62 40-102 231 160-366
CI=Confidence Interval

Figure 3

Regression analysis of the relationship between % mortality of Aedes aegypti

larvaeand concentration (ppm). Each point represents the mean of three
replicatessimultaneous with the controls

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Moribund larvae predominated during 48-hr post treatment for Citrus

microcarpa and Nephelium lappaceum and were usually deceiving unless
tapped. As mentioned, only Citrus microcarpa demonstrated sufficient toxicity
after 24 hour of treatment (80%, N=12 mortality) besides the positive control
(Black pepper). Moribund larvae on the other hand are excluded in the mortality
records. Meanwhile, some emergence of adults ensues after 72 hours of
treatment. This failure to inhibit adult emergence, in addition to larvicide mortality
suggest the nontoxicity of the extracts (all plants except black pepper) against
the pupa stage of Aedes aegypti. On the other hand, dead and moribund larvae
fail to pupate and emerged as adults. This aids in the enumeration of deaths and
survivors, thus reducing the risk of miscounting. It also serves as a reliable
control for inspecting uniformity in larval age. Percentage of adult emergence
was not recorded.

Regression analysis showed a statistically significant relationship

(p<0.01) between the mortality rates of the third-instar Aedes aegypti larvae and
ethanolic extract concentrations of the five plants (treated and positive control)
(figure 3). The fitted regression models for C. microcarpa and J. sambac were:
larval mortality = 12.4x + 1.7 X extract concentration in ppm and 1.9x – 1.566
respectively. Both have same R2-values of 0.99 indicated that the fitted model
explains 99% of the variability in larval mortality. Contrarily, for Chromolaena
odorata and Nephelium lappaceum, R2-values of 0.854, 0.878 respectively
indicate that only 85.4 and 97.8 % of the variability can be explained by the
model (Figure 3). The remaining 14.6 and 2.2% might be inherent in nature i.e.
larvae vitality is not uniform or may be caused by lurking variables such as
contaminated applicator sticks or water temperature which were not standardized
prior to the study. Suspicion is high that these variables could be random in
nature. Thus, the researchers advise the standardization of such variables
should one opt to conduct a similar study. It is important to note that while high
R2-value indicates minimum interference from variables, this does not
underestimates their influence or rules them out entirely.

Probit Analysis

A p-value of <0.05 (Chi-square=28.652) was derived from the log-probit

transformed curve of Citrus microcarpa suggesting the existence of statistically
significant relationship between its ethanolic extract concentration and larval
mortality (Figure 4).

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Figure 4
Probit-transformed responses curve of Citrus microcarpa and Piper nigrum
The lethal concentrations (LC50 and LC 90) for Citrus microcarpa and
Piper nigrum were derived from the log-probit transformed curve shown in figure
4 (Table 3). Their bioequivalence proved the notable claim regarding P. nigrum’s
larvicidal potential. If 200ppm was used as a basis, it will only take 124 mg/l for it
to kill 50% of the larvae and only 406 mg/l to eradicate the whole test population.
For Chromolaena odorata, Jasminum sambac and Nephelium lappaceum, LC50
and LC90 were no longer derived since their larval mortalities were below 50%
post-24 hour exposure. This may either imply that these plants have in reality no
larvicidal activity against Aedes aegypti 3rd-instar larvae or the LC50 too high to
be derived. In case the latter proposition is true, we encourage the retesting of
these three plants using higher increments. Thus, for plants exhibiting no to low
larvicidal activity, it would be pointless and unwise to further ascertain the toxicity
profile.

The fitted regression models for the corresponding increments (100, 200,
and 500 ppm) were: larval mortality = 7.5736x – 16.29, 12.396x – 23.5 and
16.161 x – 25.6 X time intervals. The correlation coefficients vary between each
concentration and tend to progress with dose. For 500ppm, as much as 94.4%
(R2=0.944) of the variability can be explained by the model while only 81.6% for
100ppm. This indicates that the influence of the extraneous factors irrelevant to
the study tends to decline as the concentration increases. Thus, for 500ppm, only
5.6% of the variables were outliers against 18.4% scattering seen in 100ppm.

Figure 5
Regression analysis depicting the relationship between time and % mortality of
Aedes aegypti third instars against different concentrations of Citrus microcarpa
ethanolic extract.

The larvicidal bioassay pointed out the dependency of larval mortality

against the concentration, type of plant and time of exposure. Overall, the
connections between these variables are linear, provided the uniformity and
standardization of the assay and variables. Statistical analyses also revealed the

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existence of confounding and misaligned variables, reflected by the regression

model and R2 values. This signified that some significant variables may have
been missed in the study (e.g. vitality of the larvae, temp of the water and
contamination). For Calamansi and black pepper, however, the confidence levels
are very high (Figure 2-4) whereas the influence of variables are most explicit for
C. odorata, J. sambac and N. lappaceum.

CONCLUSION

The findings of this study provide the preliminary report of the four
Philippine plants larvicidal activity against Aedes aegypti larvae. For C.
microcarpa, regression analysis showed that 50% and 90% mortality can be
attained at 581 and 1009 mg/l respectively (200ppm; CI=95%). Thus, the plant
extract may be considered as a possible larvicide substitute to Aedes aegypti if
other biological means are not available. Although it lags behind black pepper
with regards to potency (table 3), studies complementing the extract with an
affordable or accessible agonist may even proved superior considering its
nontoxicity to environment. This conforms to the study establishing the genus
Citrus (family: Rutaceae) larvicidal activity, with the monoterpene Tepinoline
noted as the bioactive compound responsible for the larvicidal property (Amusan
et.al 2005). These findings is of significant contribution to the Philippines,
especially that studies outlining the larvicidal toxicity of Philippine plants are still
currently limited, despite being a hotspot for floral diversity. The larvicidal profiles
of the tested plants may also be used to predict the larvicidal activity of the
experimented plants. The model of this study to devise greener approach could
be of interest in view of the fact that resistance and health hazards against man-
made insecticides are on the increase.

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